Sandbox 50

 =Trypsin= Trypsin is a medium sized, globular, digestive serine protease that is synthesized by the pancreas and secreted into the duodenum of the small intestine . Trypsin hydrolyzes peptide bonds based on side chain specificities of the amino acids surrounding the bond to be cleaved. Trypsin's specificity is for the positively charged side chains of lysine and arginine.

Trypsin was first named by Kuhne in 1876, when discovered trypsin differed from pepsin in its proteolytic activity at different optimal pHs. The optimal pH for trypsin is 7.5-8.5. In 1974, trypsin's three dimensional strucutre was determined, and in the late 1980s and early 1990s, site-directed mutagenesis was performed on recombinant trypsin to determine the role of specific amino acids.

Structure
Trypsin's primary structure is a polypeptide chain of amino acids. These amino acids interact with each other mostly through hydrogen bonding to form trypsin's secondary structural units. Trypsin has many important secondary structural elements, including two alpha helices (blue), an anti-parallel beta sheet (green), and random coils (gray). The arrows on these elements point toward the carboxy terminus of the protein. These secondary structures interact together to form the fully folded, native trypsin.

Polar and Nonpolar Residues
Trypsin's distribution of polar and nonpolar residues follow the rules of the hydrophobic effect. The nonpolar (gray) residues are located on the interior of the protein so they can be shielded from water, while the polar (purple) residues are distributed on the exterior of the protein because they can interact with water. This spacefill model shows the distribution of the hydrophilic and hydrophobic residues and the actual space they occupy. Again the hydrophobic, nonpolar residues are shown in gray, and the hydrophilic, polar residues are purple. This type of residue distribution in trypsin is entropically favorable becuase the water surrounding the protein does not become ordered. In this figure the polar residue interaction with water can be seen. The puprle polar residues are the residues that are interacting with the red water molecules.

Disulfide Bonds
Trypsin contains three disulfide bonds involving six cysteine residues. These disulfide bonds are intramolecular forces that stabalize the tertiary structure of Trypsin . The figure shows the yellow disulfide bonds between the cysteine residues connecting two random coils, connecting one of the alpha helices to the beta sheet, and the other disulfide connecting the two alpha helices.

Residue Charge
This charge figure shows the different charges of the amino acid residues that make up Trypsin. The blue residues have cationic side chains, the red residues have anionic side chains, the light purple are the polar, uncharged residues, and the gray residues are hydrophobic. When compared to the spacefilled figure above, the direct correlation between polarity of the side chain and charge of the side chain can be seen. Those residues with charged (blue and red) side chains as well as the polar, uncharged residues are the residues on the exterior of the protein, while the hydrophobic residues remain at the protein's core. Those residues that are cationic and anionic are able to participate in salt bridges.

Ion Contacts
Trypsin interacts with four ions. The red and yellow atoms are the ions. The yellow atoms are sulfur and the red atoms are oxygen.

Active Site
Trypsin's active site is composed of its catalytic triad, three amino acid residues that are crucial to the enzymes proteolytic function. The catalytic triad consists of Asp 102, His 57, and Ser 195 . Serine is the major player in the cleaveage of the peptide bond, thus the name serine protease. His 57 aids in the cleavage of the peptide bond through hydrogen bonds, and Asp 102 aids in the cleavage by electrostatically stabalizing the positively charged form of His 57 in the transition state. Ser 195 performs a nucleophilic attack on the substrate's peptide carbonyl. This causes the oxyanion hole to form. The formation of the oxyanion hole is stabalizing because the carbonyl oxygen that has accepted electrons during the nucleophilic attack fits nicely into this hole, and is stabalized by hydrogen bonds to the backbone NH groups of Gly 193 and Ser 195 . A figure of the oxyanion hole can be seen in greater detail in the thumbnail on the left.

The nucleophilic attack by the oxygen of Ser 195 also forms a tetrahedral intermediate. By reconstruction of the carbonyl double bound, the amino portion of the peptide leaves as a product, and an acyl-enzyme intermediate is left in the active site. Now the active site needs to be regenerated. To do this a water molecule nucleophillically attacks the carbonyl carbon, forming another tetrahedral intermediate and reforming the oxyanion hole. The nitrogen of the His 57 ring makes the oxygen of the water more nucleophilic by hydrogen bonding to one of water's hydrogens. By reforming the double bond of the carbonyl carbon, the carboxy end of the original substrate's peptide bond is released, and the active site has been regenerated. The picture in the thumbnail to the left shows the entire catalytic mechanism for a serine protease.

Trypsinogen
A zymogen is an inactive enzyme precursor. Trypsinogen is the zymogen of Trypsin that is secreted by the pancreas and is converted into the active form Trypsin in the duodenum of the small intestine. It is converted into Trypsin by proteolysis. Trypsin needs to be synthesized and secreted in an inactive form to prevent unwanted destruction of other cellular proteins, and also to regulare when and where enzyme activity of Trypsin can occur.

In order for trypsinogen to be converted to trypsin, a pro-peptide must be cleaved from trypsinogen. The image at the left shows which sequence is removed from trypsinogen, and the active site of trypsin becomes accessible for its protein substrate.

The rate of conversion of trypsinogen to trypsin can be enhanced with calcium ions. Both trypsinogen and trypsin contain an autolysis loop. This autolysis loop is comprised of residues 143-151, and contains a high affinity calcium ion binding site that is required for stability. When the calcium ion is not present, autolysis (self-digestion) will occur .

Trypsin Inhibition
Trypsin inhibitors are generally known as serine protease inhibitors or serpins. Serpins act as competitive inhibitors so they bind to the trypsin active site, rendering the enzyme inactive. There are four natural sources of trypsin inhibitors -- bovine pancreas, ovomucoid, soybeans, and lima beans. Each of these natural sources of inhibition work in different ways. Inhibitors from soybeans and lima beans inactivate insect proteases, acting as a feeding deterrent. Inhibitors from soybeans have also been found to cause pancreatic hypertrophy in rats, also acting as a feeding deterrent in this case. Other trypsin inhibitors include Ag+, benzamidine, ethylenediaminetetraacetic acid EDTA, and diisopropylfluorophosphate (DFP).

When Control Fails
When the balance between trypsin and its inhibitors, or of the activation of trypsin from trypsinogen is disturbed, pancreatitis can result. When trypsinogen is converted to trypsin early (i.e. in the pancrease), it causes the autodigestion of the pancreas. Those with cystic fibrosis often suffer from pancreatitis because it is thought that cystic fibrosis interferes with the negative feedback loop that regulates pancreatic trypsinogen secretions. When there is a high concentration of trypsin in the duodenum, a signal is sent back to the pancrease to reduce the production of trypsinogen. A high concentration of inhibitors induces pancreatic trypsinogen secretion .